It is known that various disease conditions cause abnormal levels of urobilinogen in urine, e.g., hemolytic and hepatic diseases, bilary obstruction and other lower and bile duct dysfunctions. It is recognized that the presence of urobilinogen at elevated level indicates an abnormal physiological state which requires further diagnostic procedures.
The standard test for detecting urobilinogen concentration in urine is the so-called "Ehrlich reaction", which utilizes an aqueous solution of p-dimethylaminobenzaldehyde and hydrochloric acid, referred to as Ehrlich's reagent. Urobilinogen is normally found in urine in small amounts e.g., 0.1 to 2 Ehrlich Units. One Ehrlich Unit is defined as one milligram (mg) of urobilinogen per deciliter of sample. [See Clinical Diagnosis by Laboratory Methods, p. 703, Davidsohn and Henry (1969)]. In the presence of urobilinogen, a complex of Ehrlich's reagent is produced, having absorption in the visible spectrum. The color produced can be various shades of reddish-brown, depending on the presence of interfering substances present in the urine, e.g., p-aminosalicylic acid, porphobilinogen and urea.
Currently there are available sophisticated biochemical systems which can be incorporated into dry, dip-and-read reagent strip devices, used in solution or suspension techniques, or in conjunction with spectrophotometrics and other read-out systems. Strips comprise a bibulous and nonbibulous strip, having at one end a carrier portion impregnated with an appropriate testing composition.
These dip-and-read reagent strips can incorporate Ehrlich's reagent, i.e., p-dimethylaminobenzaldehyde and hydrochloric acid. The strip is dipped into urine, and the color developed is then compared with a standard color chart prepared for use in conjunction with the reagent strip. A negative or positive for urobilinogen is obtained; if positive the approximate amount of urobilinogen present, expressed in Ehrlich Units, can be determined by comparing to a printed color chart as described hereinafter.
In carrying out testing of urine samples for urobilinogen, whether by dip-and-read strips or other techniques, it is necessary to use control solutions which are capable of producing the same color reaction produced by the presence of urobilinogen. Such controls are useful in checking the instrument used, or if the test involves human visual observation, in checking the skill of the technician, i.e., as an "unknown". In addition, controls are useful for educational purposes, e.g., in instructing technicians in carrying out urobilinogen tests.
In order to be most useful, especially in conducting "blind" skill tests, urobilinogen controls must not only closely match the color produced by the presence of urobilinogen in urine, but also must not have olfactory properties noticeably different from urine olfactory properties, and must be light-stable for at least 4 hours and preferably for 48 hours.
It is common that such controls involve the use of the substance which is being tested; however, because urobilinogen is unstable and generally unavailable, other chemical compounds have been used as a control material. Compounds which have been used as a urobilinogen control are indoles. Although it has been known that indoles, freshly prepared, do react with Ehrlich's reagent to produce reddish-brown colors that closely resemble the colors formed by urobilinogen present in urine and Ehrlich's reagent, indoles suffer the disadvantage of possessing a characteristic, highly noticable unpleasant odor; are relatively insoluble in aqueous solutions; are lightsensitive and are very unstable.
The present invention provides a method and improved indole composition for use as a urobilinogen control standard which overcomes these problems.